Name: intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes
Uploaded: 2018-03-15T13:00:00
Description: Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Gli esperimenti sono stati supportati da Marga e Walter Boll-Stiftung, Kerpen, Germania; Progetto 210-02-16 (Manno), progetto 210-03-15 (MAAA) e dalla Fondazione di ricerca tedesca (DFG; AL1632-02; MAAA), Bonn, Germania; Centro di medicina molecolare Colonia (CMMC; Ospedale universitario di Colonia; Programma di avanzamento di carriera; MAAA), Fortuna Köln (facoltà di medicina, Università di Colonia; KD).

Tutte le procedure di animali sono stati condotti in conformità a protocolli approvati dalle autorità di governo locale (Land NRW, AZ: 2012.A424) ed erano in conformità con la legge tedesca sul benessere degli animali e le norme sul benessere degli animali utilizzati per esperimenti o per altri fini scientifici. Poiché analisi di funzione polmonare possono influenzare la struttura del polmone e quindi successive analisi istologica, la misurazione di Res e CDIN e la preparazione e la fissazione del polmone per istomor...

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Questo rapporto fornisce tre protocolli per tre diversi metodi analizzare l'impatto dell'obesità sul metabolismo del glucosio e gli esiti polmonari. In primo luogo, il test di tolleranza al glucosio offre la possibilità di analizzare l'assorbimento intracellulare del glucosio e può essere indicativo di insulino-resistenza. In secondo luogo, pletismografia di tutto il corpo è una tecnica per misurare la funzione polmonare ed è quindi utile per testare l'efficacia di nuovi trattamenti. In terzo luogo, un protocollo st...

Secondo il mondo salute organizzazione (OMS) nel 2008, più di 1,4 miliardi adulti, di età compresa tra 20 e più anziani, erano in sovrappeso con un indice di massa corporea (BMI) maggiore o uguale a 25; ulteriormente, oltre 200 milioni di uomini e donne quasi 300 milioni erano obesi (BMI≥30)1. Obesità e sindrome metabolica sono fattori di rischio importanti per un gran numero di malattie. Mentre l'obesità e il tessuto adiposo aumentato concomitante bianco massa è stato intimamente collegato per tipo 2 diabete2,3, malattie cardiovascolari, tra cui la malattia coronarica (CHD), insufficienza cardiaca (IC), fibrillazione atriale4 e osteoartrite5, loro ruoli funzionali nella patogenesi dei disordini respiratori rimangono poco compresi. Tuttavia, gli studi epidemiologici hanno dimostrato che l'obesità è fortemente associata con condizioni respiratorie croniche, tra cui dispnea da sforzo, sindrome di apnea ostruttiva del sonno (OSAS), sindrome di hypoventilation di obesità (OHS), cronica malattia polmonare ostruttiva (BPCO), embolia polmonare, polmonite da aspirazione e asma bronchiale6,7,8,9. Potenziali meccanismi che collegano l'obesità e metabolismo perturbato, per esempio, l'insulino-resistenza e diabete di tipo II, alla patogenesi dell'affezione polmonare cronica comprendono non solo le conseguenze fisiche e meccaniche del peso ma anche di guadagno sulla ventilazione indurre una condizione infiammatoria subacuta cronica10,11. L'aumento dell'obesità e malattie polmonari durante l'ultimo decennio, accoppiati con la mancanza di efficaci strategie preventive e approcci terapeutici, evidenzia la necessità di studiare i meccanismi molecolari per definire nuove vie per gestire l'obesità-collegata del polmone malattie.
Qui, descriviamo tre prove standard, che sono basi importanti per studiare l'obesità e il suo impatto sulla struttura del polmone e sulla funzione in modelli murini: (1) intraperitoneale glucosio tolleranza (ipGTT) (2) misurazione della resistenza delle vie aeree (Res) e delle vie respiratorie conformità del sistema (Cdin); e (3) preparazione e la fissazione del polmone per la successiva valutazione istologica quantitativa. Il ipGTT è un test di screening robusto per l'assorbimento del glucosio di misura e quindi l'effetto dell'obesità sul metabolismo. La semplicità del metodo consente buona standardizzazione e pertanto la comparabilità dei risultati tra i laboratori. Metodi più sofisticati, come hyperglycemic morsetti o studi sugli isolotti isolati, possono essere utilizzati per un'analisi dettagliata del fenotipo metabolico12. Qui valutiamo la tolleranza al glucosio per definire uno stato di disordine sistemico e metaboliche associate all'obesità come base per ulteriori studi su un risultato polmonare. Per valutare l'effetto dell'obesità e del disordine metabolico sulla funzione polmonare, abbiamo misurato la resistenza delle vie aeree (Res) e la conformità del sistema respiratorio (Cdin). Per caratterizzare l'affezione polmonare, sono disponibili metodi sfrenati come pure trattenuti per la valutazione della funzione polmonare. Pletismografia sfrenato in animali liberi di muoversi imita uno stato naturale, che riflette i modelli di respirazione; al contrario, metodi invasivi, come misura di impedenza di ingresso di Res e CDIN in topi anestetizzati profondamente per valutare la meccanica polmonare dinamica, sono più accurate13. Poiché le condizioni respiratorie croniche sono riflessi da alterazioni istologiche del tessuto polmonare, fissazione del polmone adeguato per un'ulteriore analisi è imminente. La scelta del metodo di fissazione del tessuto e preparazione dipende il vano del polmone che sarà studiato, ad esempio, lo svolgimento di vie respiratorie o del polmone parenchima14. Qui, descriviamo un metodo che permette la valutazione qualitativa e quantitativa delle vie aeree conduce per studiare l'effetto dell'obesità su sviluppo di asma.

<table>
	<tbody>
		<tr>
			<td>GlucoMen LX</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>38969</td>
			<td>blood glucose meter</td>
		</tr>
		<tr>
			<td>GlucoMen LX Sensor</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>39765</td>
			<td>Test stripes</td>
		</tr>
		<tr>
			<td>Glucose 20%</td>
			<td>B. Braun, Melsung, Germany</td>
			<td>2356746</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe Software</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-002</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe RC Single Site Mouse Table</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-001</td>
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			<td>FPRC Controller</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1075-001</td>
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			<td>FPRC Aerosol Block</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1106-001</td>
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			<td>Aerogen neb head-5.2-4um</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
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			<td>Blunt scissors</td>
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intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes

L'obesità e i disturbi respiratori sono gravi problemi di salute. L'obesità sta diventando un'epidemia emergente con un numero previsto di oltre 1 miliardo individui obesi in tutto il mondo entro il 2030, rappresentando così un crescente onere socio-economico. Contemporaneamente, comorbidità associate all'obesità, compreso il diabete così come cuore e malattie polmonari croniche, sono continuamente in aumento. Anche se l'obesità è stata associata con un rischio aumentato per le esacerbazioni di asma, peggioramento dei sintomi respiratori e scarso controllo, il ruolo funzionale di obesità e metabolismo perturbato nella patogenesi della malattia polmonare cronica è spesso sottovalutato, e meccanismi molecolari rimangono evasivi. Questo articolo si propone di presentare i metodi per valutare l'effetto dell'obesità sul metabolismo, così come la struttura del polmone e la funzione. Qui, descriviamo tre tecniche per studi di topi: (1) valutazione della tolleranza al glucosio intraperitoneale (ipGTT) per analizzare l'effetto dell'obesità sul metabolismo del glucosio; (2) misurazione della resistenza delle vie aeree (Res) e la conformità del sistema respiratorio (Cdin) per analizzare l'effetto dell'obesità sulla funzione polmonare; e (3) preparazione e la fissazione del polmone per la successiva valutazione istologica quantitativa. Malattie polmonari legate all'obesità sono probabilmente multifattoriali, derivanti dalla disregolazione infiammatori e metabolici sistemici che potenzialmente compromettere la funzione polmonare e la risposta alla terapia. Pertanto, una metodologia standardizzata per studiare i meccanismi molecolari e l'effetto di nuovi trattamenti è essenziale.

Risultati rappresentativi del test di tolleranza al glucosio intraperitoneale (ipGTT) (Figura 4), polmone funzione test (Figura 5) e rappresentante immagini che illustrano ematossilina ed eosina macchiato polmoni (Figura 6).
Il ipGTT è stato effettuato in topi obesi (blu) dopo 7 settimane di dieta-alta-grassi (HFD). Standard topi di dieta-...

Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

L'incidenza dell'obesità è in aumento e aumenta il rischio di malattie polmonari croniche. Per stabilire i meccanismi sottostanti e le strategie preventive, animale ben definiti modelli sono necessari. Qui, mettiamo a disposizione tre metodi (prova di tolleranza di glucosio, pletismografia corporea e fissazione del polmone) per studiare l'effetto dell'obesità sui risultati polmonari nei topi.

Prova di tolleranza di glucosio intraperitoneale, misura della funzione polmonare e la fissazione del polmone per studiare l'impatto dell'obesità e metabolismo alterato sugli esiti polmonare

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

The overall goal of the following three methods is to study the impact of obesity and metabolic disorders on lung structure and function. These methods can help to answer key questions in the respiratory research field about the impact of metabolic disorders on lung structure and lung function. The advantage of these techniques is that they can be combined to assess the functional role of metabolism on the pathogenesis of chronic lung diseases and to test different therapeutical approaches.The implications of these techniques extend beyond phenotyping to what's elucidating the specific functional role of metabolic targets in the pathogenesis of lung diseases. These methods can also be applied to the study of structure and function in both healthy animals with genetic modifications but also in animal models of COPD and asthma. Generally, individuals new to these methods will struggle as insertion of the tracheal tube, the insufflation of the lung and the administration of the deep anesthesia, they all require experience and training.We first had the idea for these methods when we discovered that early onset obesity in mice but also in humans were intimately linked to impact metabolism and to chronic lung diseases beyond infancy. To measure intraperitoneal glucose tolerance, gently restrain a 12-hour fasted adult mouse by the tail and use sterile scissors to incise the tail tip. Immediately apply a free-flowing drop of blood to the test strip of the blood glucose meter to measure the fasted blood glucose level.The test result will appear on the glucometer screen after four seconds. After measuring the glucose for each animal, label them individually with colored markers and weigh the animals. Then use a 27-gauge needle attached to a one-milliliter syringe to administer 100 microliters of glucose per ton grams of body weight to each animal via intraperitoneal injection and measure the blood glucose levels 15, 30, 60 and 120 minutes later with new test strips as just demonstrated.To assess the lung function, place an adult mouse in the supine position on a pad and apply ointment to the animal's eyes. After confirming a lack of response to toe pinch, wet the fur in the thyroid region with 70%ethanol and use forceps to lift the skin at the midline between the jugular notch at the sternum and the tuber symphysis of the mentum. Using blunt scissors, carefully make a one-centimeter skin incision in the tented skin to visualize the underlying subcutaneous adipose tissue and thyroid gland and expose the trachea with careful blunt separation of both thyroid lobes at the isthmus and dissection of the sternothyroid and sternothyroid muscles.Using sharp forceps, pass a 4-0 braided surgical suture between the trachea and the esophagus and carefully incise the trachea close the the larynx between the tracheal cartilages with microscissors. Intubate with a tracheal tube securing the tube with a surgical suture and move the animal to the heated bed of a body chamber. Connect the tracheal tube to the face plate and turn on the ventilator.Confirm movement of the thorax contemporaneously with the ventilation rate to confirm proper placement of the tracheal tube and watch the pressure signal on the computer screen. To control changes in the transpulmonary pressure during ventilation, insert an esophageal tube to the level of the lungs watching the screen to facilitate placement of the tube to where maximal pressure deflection and minimal heart artifacts can be observed. Begin the data acquisition according to the manufacturer's instructions and apply 10 microliters of PBS on the nebulizer.Start nebulization after five minutes of acclamation followed by a response phase of three minutes during which the airway resistance and respiratory system compliance are measured. Allow the animal to recover for three minutes before starting the next nebulization and follow the software instructions for stepwise application of increasing 10-microliter concentrations of methacholine on the ventilator. For quantitative histomorphometric analysis of the lung tissue, use blunt scissors to make a small incision in the diaphragm of an adult mouse and use curved blunt scissors to make a peristernal incision along the entire length of the rib cage to gently open the thorax.Lift the rib cage to expose the pleural cavity and remove the thymus. After removing the heart, use sharp forceps to prepare and expose the trachea in order to pass a 4-0 braided surgical suture between the trachea and the esophagus followed by careful incision of the trachea close to the larynx between the tracheal cartilages. Intubate with a 26-gauge intravenous cannula and use a fixative agent to inflate the lungs by pressure fixation at a constant pressure of 20 centimeters of water.After 30 minutes at room temperature, ligate the trachea and remove the cannula at the same time. Then carefully excise the lungs without damaging the tissue and store the lungs in fresh fixative agent at four degrees Celsius overnight. Obese mice demonstrate an increase in serum glucose levels 15 and 30 minutes after intraperitoneal glucose injection indicating an impaired cellular glucose uptake compared to standard diet fed mice.Invasive lung function analysis shows an up to 1.5-fold increase of airway resistance in obese, high-fat diet fed mice after stimulation with methacholine compared to control animals. Hematoxylin and eosin staining of lung parenchyma sections after intratracheal installation reveals multiple uninflated areas, thick alveolar septa and polygonal-shaped alveoli in lungs instilled with too little pressure. In contrast, too much instillation pressure leads to overinflated areas with destructed alveolar septa.Application of the appropriate amount of pressure during lung fixation however leads to a completely inflated lung with round-shaped alveoli. Once mastered, the intraperitoneal glucose tolerance tests can be completed in two and a half hour. The lung function tests can be completed in one hour, and the lung insufflation can be completed in under an hour.While attempting this procedure, it's important to remember that adapting the mice, training the investigator and working in a quiet and stress-reduced environment all are recommended for optimal data acquisition. After its development, this technique paved the way for researchers in the fields of metabolism and respiratory diseases to explore the mechanisms involved in and potention of treatment strategies of chronic lung disease using small animal models. After watching this video, you should have a good understanding how to investigate the impact of obesity and metabolic disorders on lung structure and function.Don't forget that working with animals or surgical instruments can cause injuries or skin reactions and that precautions such as wearing the appropriate personal protection equipment should always be taken while performing these procedures.

Research

JoVE Journal

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